Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

Abstract

Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes

“…As a means of further understanding the role of MsSPL8 in plant development and abiotic stress response, and concomitantly generating transgene-free germplasm with reduced MsSPL8 activity, we utilized CRISPR/Cas9 technology to simultaneously edit multiple MsSPL8 alleles in alfalfa. Although the CRISPR/Cas9 platform has proven extremely effective in many plant species to date, including those with polyploid genomes ( Morineau et al, 2017 ; Liu et al, 2018 ; Huang et al, 2020 ), it has only very recently begun to gain traction in alfalfa ( Gao et al, 2018b ; Chen et al, 2020 ; Wolabu et al, 2020 ; Bottero et al, 2021 ; Curtin et al, 2021 ). The first attempt at utilizing this technique in this species involved the targeting of another SPL gene ( MsSPL9 ), and while successful edits at the target site were detected, they occurred with extremely low frequencies of up to approximately 2.2% of alleles within the tissue tested ( Gao et al, 2018b ).…”

“…However, subsequent studies have led to substantially higher editing frequencies and consequent phenotypic alterations in alfalfa, with up to 100% allelic mutation frequency observed in the first generation when targeting the M. sativa stay-green ( MsSGR ), phytoene desaturase ( MsPDS ) and PALM1 genes ( Chen et al, 2020 ). While the use of a multiplex gRNA CRISPR/Cas9 system was required to achieve high editing frequencies in alfalfa in certain instances ( Wolabu et al, 2020 ; Bottero et al, 2021 ), this has not always been the case ( Chen et al, 2020 ). In the current study, we were able to achieve high frequencies of indels in MsSPL8 alleles in the first generation (with up to three of four alleles mutated) with a single gRNA ( Figures 2B , 3B,C ), and in a manner that was homogeneous in at least a proportion of genotypes ( Figure 2C ).…”

Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

“…Because of the extreme heterozygosity of alfalfa, an individual plant (e.g., regenerative clone C23) normally possesses four different alleles of the same locus, and it is thus possible to determine the exact localization of a mutation by segregation analysis ( Bottero et al, 2021 ). Bioinformatic analysis of the Tnt1 -flanking sequences of the mfl mutant plant and its progeny showed that the mfl mutation colocalized with the MsNAC39 gene in chromosome 3.4 ( Figure 1I ).…”

“…In the last years, the increased efficiency of the CRISPR/Cas9 system in highly regenerative alfalfa germplasms has allowed full allelic knockout of an individual gene in the T0 generation ( Chen et al, 2020 ; Wolabu et al, 2020 ; Bottero et al, 2021 ). Naturally, this optimized system can help to validate in alfalfa the strategies found in the model species Medicago truncatula .…”

“…As an example of this transfer, null palm1 mutants produce pentafoliate leaves rather than wild-type trifoliate leaves in both M. truncatula ( Chen et al, 2010 ) and alfalfa ( Chen et al, 2020 ). Although the high expression of this type of beneficial traits (e.g., multifoliated leaves) obtained by the full allelic knockout of an individual alfalfa gene (e.g., palm1 ) in elite germplasms is not impossible, it requires an adaptation of the current introgression processes, a fact that increases the cost and time of production of a genetically modified alfalfa cultivar ( Bottero et al, 2021 ).…”